Please cite this article as: Wang, X.-C., Chen, L., Ma, C.-L., Yao, M.-Z., & Yang, Y.-J., Genotypic variation of beta-carotene and lutein contents in tea germplasms, Camellia sinensis (L.) O. Kuntze, Journal of Food Composition and Analysis (2008), doi:10.1016/j.jfca.2009.01.016 This is a PDF ?le of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its ?nal form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Tea is the most popular non-alcoholic healthy beverage in the world, and its water-soluble components such as polyphenols hold important benefits for human health. The lipid-soluble components such as beta-carotene and lutein, however, have not yet been utilized. In order to assess the possibility in using tea leaves as a new source of natural carotenoids, beta-carotene and lutein contents were estimated in new spring shoots of different genotypes and leaf positions of tea plant (Camellia sinensis) using a reverse-phase HPLC approach. There was no significant difference among the three assessed tea varieties (C. sinensis var. sinensis, var. assamica and var. pubilimba) from four different countries (p=0.549 and 0.092, respectively). But significant differences were found among different leaf positions (maturity). The highest levels of beta-carotene (343.2 mg/kg) and lutein (232.0 mg/kg) were found in C. sinensis var.1

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E-mail address: liangchen@mail.tricaas.com (Dr L. Chiang).

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Academy of Agricultural Sciences; National Center for Tea Improvement of China, 9 South

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Research Center for Tea Germplasm and Improvement, Tea Research Institute Chinese

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assamica and, highest coefficient of variation was present in C. sinensis var. sinensis (44.3%). The highest level of beta-carotene content and lutein contents were 42.1 and 16.3 times higher than the lowest among the studied 119 germplasms, respectively. Furthermore, contents of beta-carotene and lutein in mature leaves were higher than young leaves and stems. The results showed that tea may have potential as a new source of carotenoids. Keywords: Tea plant; (Camellia sinensis); Beta-carotene; Germplasm; Lutein; Food analysis;

1 Introduction

and algae. Beta-carotene and lutein are two nutritionally important plant-derived carotenoids.

and tissue. It has the highest provitamin A activity, and deficiency of this compound can result in xerophthalmia, blindness, and premature death (Mayne, 1996). It was estimated that 250 million preschool children worldwide are deficient in vitamin A

A nutrition could prevent 1.2 million deaths annually among children aged 1-4 years (West et al., 1989). In order to solve this problem, methods such as genetic modification to increase beta-carotene content have been used (Ye et al., 2000). Beta-carotene is also beneficial in the prevention of cancer, improvement of antioxidant status and immune response, anti-radiation effects, protection against photo-induced tissue damage, etc. (Bendich & Olson, 1989; Krinsky & Johnson, 2005). In market, the product of beta-carotene contains synthetic and natural forms. The synthetic form is all-trans form. The naturally occurring form contains about 50% trans-isomer and several other forms, the most prevalent being 9-cis-beta-carotene (Ben-Amotz & Levy, 1996; Yeum et al., 1995). The 9-cis form of beta-carotene has much

Beta-carotene is the most widely studied of the major carotenoids in the human diet, blood

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Carotenoids are a large group of lipid-soluble pigments synthesized in plants, bacteria, fungi

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Food composition

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greater antioxidant activity in humans than the synthetic all-trans form (Ben-Amotz & Levy, 1996). Moreover, in vitro (Toba et al., 1997) and preliminary human data indicate that the naturally occurring forms of beta-carotene have a better ability to block malignant transformation than the synthetic all trans-isomer (Yeum et al., 1995). So, the natural form is better than the synthetic form. Lutein has no provitamin A activity in humans, but it has biological activities that have

Bone, 2001). Lutein and its isomer, zeaxanthin (Humphries & Khachik, 2003), play very

associations between higher intake or serum levels of lutein and lower risk for developing

degeneration (Shao, 2001; Granado et al., 2003; Johnson, 2004). The market values of betacarotene and lutein were estimated at US$242 million and US$139 million in 2004, respectively (http://www.the-infoshop.com/study/bc31318-carotenoids.html). Tea is the most popular non-alcoholic beverage in the world. It has socio-economic and cultural importance for some Asian, African and South American countries, such as China, India, Japan, Sri Lanka and Kenya. Tea plants, which originated from southwestern China, Yunnan province (Yu, 1986), including 5 species and 2 varieties (Chen et al., 2000), belonged to Theaceace, genus Camellia L., Sect. Thea (L.) Dyer. Usually one species and two varieties, i.e. C. sinensis (L.) O. Kuntze, C. sinensis var. assamica (Masters) Kitamura and C. sinensis var. pubilimba Chang are cultivated worldwide (Chen et al., 2006a, b). The China National Germplasm Hangzhou Tea Repository (CNGHTR) in the Tea Research Institute Chinese Academy of Agricultural Sciences (TRICAAS for short) had by the end of 2006 preserved about 2,665 accessions of tea germplasms (Chen et al., 2006a). As a health beverage, the3

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cardiovascular disease, several types of cancers, cataracts and age-related macular

human retina, corresponding to about 36% of the total (Sommerburg et al., 1999; Landrum &

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attracted great attention in relation to human health. It is the dominant carotenoid in the

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medicinal effects of tea have a long and rich history. The main components of tea are polyphenols (mainly catechins—about 10% of dry weight), caffeine (about 4% of dry weight), and theanine (about 1% of dry weight) (Yamamoto et al., 1998). Long-term drinking of tea may protect against several forms of cancers, cardiovascular diseases, the formation of kidney stones, bacterial infections, and dental cavities (Trevisanato & Kim, 2000; Wheeler & Wheeler, 2004; Yang et al., 2007; Katiyara et al., 2007). In earlier studies, scientists have focused mainly on tea polyphenols and other water-soluble components, but in tea leaves there are some important lipid-soluble components, such as carotenoids including betacarotene and lutein, which have rarely been investigated up until now. Beta-carotene and lutein are the main carotenoids in tea leaves (Shao, 2003). Actually, compared with other crops, contents of beta-carotene and lutein in tea leaves are high (Wang et al., 2004). However, there is very limited information about genotypic and spatial differences of betacarotene and lutein contents in tea plant. In order to assess the possibility of making full use

tea germplasms at the same growth environment, (2) to investigate the variability in betacarotene and lutein contents in tea plants originated from different countries and, (3) to determine the spatial variability in beta-carotene and lutein contents on shoots and mature

analyze the genotypic variability in beta-carotene and lutein contents in different varieties of

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of tea leaves as new source of natural carotenoids, the objectives of this study were (1) to

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accessions were cultivated in the same environment with same agronomical measurements in the CNGHTR at Hangzhou, China. In order to screen genotypic variation of beta-carotene and lutein contents in tea germplasms and understand the variation in different positional leaves on young shoots, ‘two and a bud’ of the first flush of the 119 accessions germplasms, as well as ‘one and a bud’, the 2nd, 3rd, 4th, 5th leaf, green stems and fully mature leaves were harvested from cultivars ‘Longjing 43’ and ‘Aifeng 20’ in spring. All the samples were fixed by microwave oven (power 1.0 KW, 2 min) immediately after being plucked, then dried with hot air at 60 °C. Samples were stored in airtight containers in the dark until they were analyzed.

According to slightly modified protocol of Tao et al. (2002), 1.0 g of sample was extracted

centrifuged at 4,000 g for 10 min. The pellet was extracted with 30 ml acetone (containing 0.1% BHT) 2~3 times as above, until it became colorless. The supernatants were combined and the acetone removed in a rotary evaporator (Buchi R-124, Flawil, Switzerland) at 35 °C and ≈0.09 Mpa. The residue was dissolved in 20 ml ether (containing 0.1% BHT) and saponified by addition of 20 ml of 10% methanolic KOH. This was followed by standing for 3 h at 4 °C in the dark. Then 20 ml of NaCl (10% w/v) was added to this mixture. After phase

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with 10 ml acetone (containing 0.1% BHT as antioxidant) with oscillator for 10 min, then

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2.2

Chemicals and standards

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separation, the upper phase was removed and the lower phase was extracted twice with 40 ml ether (containing 0.1% BHT). The combined ether phases were rinsed with small volumes of water until the pH was reduced to 7.0. The combined ether phases were dried by anhydrous Na2SO4 and reduced in the rotary evaporator at 35 °C and ≈0.09 Mpa. The residue was disolved in 5 ml of methanol / ethyl acetate (1:1, containing 0.1% BHT) and passed through a 0.45 μm membrane filter before further analysis.

MA, USA), with auto-injector and UV-visible diode array detector. The column was a

Ltd, Dalian, China), which was protected by a guard column containing the same stationary

was 20 μl. Peaks were identified by comparing their retention times with standards. Figures 1 and 2 indicate the chromatograms of the reference mixture and a tea sample, respectively. Quantification was carried out using the external standard method; standard curves were calculated by linear regression analyses. The fitted lines showed very good correlations coefficients of R2=0.995 and R2=0.994 for beta-carotene and lutein, respectively. Recovery experiments were repeated 5 times. The samples were spiked with known amounts of the standards during the extraction. The mean values for the recoveries were 99.2% and 87.4% for beta-carotene and lutein, respectively. [Figs. 1-2 here]6

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wavelength was 450 nm. The column was maintained at 30 °C. The sample injection volume

Separation and analysis of carotenoids were carried out by a high performance liquid

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2.4

Data processing

Analysis of tea germplasms was performed once, and different positional samples were analyzed in triplicates and averaged. The analyses were processed using software SAS ver. 8.02 (SAS Institute Inc., Cary, NC, USA).

3 Results and discussions3.1

carotene and lutein in new shoots among 3 varieties of tea plants are summarized in Tables 1,

found in C. sinensis var. assamica, the highest CV was found in C. sinensis var. sinensis (44.3%). There was no significant difference among three varieties (p=0.549 and 0.092, respectively). [Tables 1, 2, and 3 about here]

presented in Table 1 showed that the beta-carotene content ranged from 17.2 mg/kg (‘Qianmei 601’) to 727.7 mg/kg (‘Yungui Daye’), with an average of 306.0 mg/kg. Lutein content in the var. sinensis germplasms ranged from 48.2 mg/kg (‘Baijiguan’) to 417.8 mg/kg (‘Yungui Daye’), with an average of 191.8 mg/kg. The highest level of beta-carotene and lutein contents were 42.1 and 8.7 times higher than the lowest, respectively. The albino mutants, ‘Tiantai Baicha’, ‘Huangjinju’ and ‘Baijiguan’, except ‘Baiye 1’, whose leaves were

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for variability among the germplasms for the contents of beta-carotene and lutein. Data

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Variability of beta-carotene and lutein contents among different germplasms

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2, and 3. The highest means of beta-carotene (343.2 mg/kg) and lutein (232.0 mg/kg) were

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The mean, range, coefficient of variation (CV) and F-test value of the contents of beta-

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Variability of beta-carotene and lutein contents among different varieties

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white in spring, had low beta-carotene and lutein contents. Similar results were found in a rice mutant (Cui et al., 2001). C. sinensis var. assamica: Nineteen germplasms of C. sinensis var. assamica were assayed for variability among the germplasms for the contents of beta-carotene and lutein. The data

to 488.5 mg/kg (‘KNY-1’), with an average of 343.2 mg/kg. Lutein content in the var.

contents were 21.6 and 5.0 times higher than the lowest, respectively.

C. sinensis var. pubilimba: The contents of beta-carotene and lutein were also analyzed in 22 C. sinensis var. pubilimba accessions. Data presented in Table 3 show that the beta-carotene content ranged from 28.9 mg/kg (‘Yuechang Baimaocha’) to 553.9 mg/kg (‘Yuebai Yuannan’), with an average of 319.9 mg/kg. Lutein content in the var. pubilimba ranged from

average of 208.9 mg/kg. The highest level of beta-carotene and lutein contents were 19.2 and 8.8 times higher than the lowest, respectively. As can be seen in Tables 1-3, the highest contents of beta-carotene (727.7 mg/kg) and lutein

mg/kg and 22.6 mg/kg), respectively. The results suggested that there were significant genotypic differences of carotenoid content among tea plant germplasms. Carotenoid content is mainly controlled by poly-gene (Graham and Rosser, 2000), so we can use some breeding approaches such as gene polymerization to raise carotenoid content of tea plant.

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(488.5 mg/kg) in tea germplasms were 42.1 and 21.6 times higher than the lowest onces (17.2

In China, a large portion of tea farmers pluck only spring shoots to produce elite teas in spring, and do not pluck in summer and autumn due to the low price of summer and autumn teas. A great number of tea leaves are left on the tea bushes and are not harvested every year. At the end of spring or early in the summer, tea bushes are often pruned to prepare a new crown for the next year and a great amount of tea leaves and young stems are generated.9

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shoots, young leaves (one and a bud, the 2nd leaf) and green stems, have lower beta-carotene

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These materials are usually wasted. Previous investigations have found that the beta-carotene content of summer and autumn tea leaves was 3 to10 times higher than the spring tea leaves (Wang et al., 2006). Our results showed that beta-carotene (mean 311.5 mg/kg) and lutein (mean 199.5 mg/kg) contents of tea plant leaves were much higher than common cereal crops, such as wheat (0.02 and 1.4mg/kg) and maize (1.7 and 4.3mg/kg), or vegetables, such as carrot (61.5 and 5.1mg/kg), Brassica vegetables (0.1~8.1 and 0.2~6.8mg/kg) and tomato (9.18 and 1.0mg/kg) (Adom et al., 2003; Kandlakunta et al., 2008; Niizu et al., 2005; Singh et al., 2007). Therefore, tea plant leaves, especially summer and autumn tea leaves and young stems, might be of potential use for extraction of carotenoids. They might also be useful for production of tea powder as a natural carotenoid supplement in diverse foods, such as cake or noodles. Thus, the utilization value of summer and autumn tea leaves and the tea farmers’ income will both be increased.

(863 Plan) (2006AA10Z171), the National Key Technologies R&D Program (2006BAD06B01) and the R&D Infrastructure and Facility Development (2005DKA2100208) of the Ministry of Science and Technology of China to Liang Chen. The authors thank

thank Prof. Dr. Zeno Apostolides in University of Pretoria, South Africa, Dr. Chen Yin in the Zhejiang Centre for Disease Control and Prevention, China, and the anonymous reviewers for their critical reviews and constructive comments.